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目的:研究急性缺血/缺氧时肝癌细胞HepG2增殖率及自噬的变化,探讨自噬的作用及机制。方法 :以Western印迹法检测缺氧诱导因子-1(hypoxia induced factor-1,HIF-1)表达并确定体外模拟模型的可靠性;吖啶橙染色后采用荧光显微镜定性观察自噬;以自噬特异性蛋白LC3及P62(P62/SQSTM1)信号蛋白的变化表明自噬的诱导和可能的调控机制;以CCK8检测3-甲基腺嘌呤(3-MA)抑制自噬前后急性缺血/缺氧下HepG2细胞的增殖率变化。结果:急性缺血/缺氧2 h后,HepG2细胞显著表达HIF-1α蛋白,表明体外急性缺血/缺氧模型的可靠性。急性缺血/缺氧可快速诱导肝癌细胞HepG2产生自噬,继而出现HepG2细胞显著增殖活跃;3-MA抑制自噬后,可特异性抑制急性缺血/缺氧所诱导的HepG2细胞增殖;急性缺血/缺氧条件下HepG2细胞P62信号蛋白表达显著下调,自噬抑制后逐渐恢复正常表达水平。结论:自噬在肝癌体外急性缺血/缺氧过程中对肿瘤起到保护作用,其机制可能与P62蛋白的清除有关;抑制自噬可显著降低肝癌细胞增殖率。自噬可能成为肝癌治疗的新靶点。
Objective: To study the proliferation and autophagy changes of hepatoma HepG2 cells during acute ischemia / hypoxia and to explore the mechanism and mechanism of autophagy. Methods: The expression of hypoxia induced factor-1 (HIF-1) was detected by Western blotting and the reliability of in vitro model was determined. Acridine orange staining was used to detect the autophagy by fluorescence microscopy. The changes of the specific proteins LC3 and P62 (P62 / SQSTM1) signaling proteins indicate the induction of autophagy and the possible regulatory mechanisms; the detection of 3-methyladenine (3-MA) by CCK8 before and after autophagy inhibition of acute ischemia / Proliferation rate of HepG2 cells. Results: After 2 h of acute ischemia / hypoxia, HIF-1α protein was significantly expressed in HepG2 cells, indicating the reliability of acute ischemia / hypoxia model in vitro. Acute ischemia / hypoxia can rapidly induce HepG2 hepatoma cell autophagy, followed by significant proliferation of HepG2 cells; 3-MA inhibition of autophagy can specifically inhibit acute ischemia / hypoxia induced HepG2 cell proliferation; acute The expression of P62 protein in HepG2 cells was significantly down-regulated under the condition of ischemia / hypoxia, and gradually restored to the normal level after autophagy inhibition. CONCLUSION: Autophagy plays a protective role in the process of acute ischemia / hypoxia in vitro and the mechanism may be related to the clearance of P62 protein. Inhibition of autophagy can significantly reduce the proliferation rate of HCC cells. Autophagy may become a new target for the treatment of liver cancer.